Iron is an essential trace element that is required for growth and development of all living organisms; it is indispensable for DNA synthesis and a broad range of metabolic processes. However, disturbances of iron metabolism have been implicated in a number of significant mammalian diseases, including, but not limited to iron deficiency anemia, hemosiderosis or the iron overload disease hemochrornatosis (Pietrangelo, A. (2002) Am J Physiol. Gastrointest. Liver Physiol. 282, G403-414; Andrews, N. C. (2000) Annu. Rev. Genomics Hum. Genet. 1, 75-98; Philpott, C. C. (2002) Hepatology 35, 993-1001; Anderson and Powell (2002) Int J Hematol 76, 203-203; Beutler et al., (2001) Drug-Metab. Dispos. 29, 495-499). Under physiological conditions, a human's iron content is regulated by controlling absorption. In mammals, iron absorption occurs predominantly in the duodenum and upper jejunum, and is the only mechanism by which iron stores are physiologically controlled (Philpott, C. C. (2002) Hepatology 35, 993-1001). Following absorption, iron is bound to circulating transferrin and delivered to tissues throughout the body. In the liver, the major site of iron storage, transferrin-bound iron is taken into the cells by receptor-mediated endocytosis via the classical transferrin receptor (TfR1) (Collawn et al. (1990) Cell 63, 1061-1072) and presumably in greater amounts via the recently identified homologous transferrin receptor 2 (TfR2) (Kawabata et al. (1999) J Biol Chem 274, 20826-20832). The extracellular domain of this protein is 45% identical to the corresponding portion of TfR1 (Id.). TfR2 can also bind diferric transferrin and facilitate the uptake of iron. Mutations in TfR2 have been associated with certain forms of hemochromatosis demonstrating the important role for TfR2 in iron homeostasis (Philpott, C. C. (2002) Hepatology 35, 993-1001; Camasehella et al., (2000) Nat. Genet. 25, 14-15; Fleming et al., (2002) Proc. Natl. Acad. Sci. USA 99, 10653-10658). TfR2 is predominantly expressed in the liver (Fleming et al., (2000) Proc. Natl. Acadi. Sci. USA 97, 2214-2219; Subramaniam et al., 2002) Cell Biochem. Biophys. 36, 235-239), however, the exact cellular localization is still unknown.
A feedback mechanism exists that enhances iron absorption in individuals who are iron deficient, whereas iron absorption is reduced in persons with iron overload (Pietrangelo, A. (2002) Am J Physiol Gastrointest Liver Physiol 282, G403-414; Philpott, C. C. (2002) Hepatology 35, 993-1001; Anderson and Powell (2002) Int J Hematol 76, 203-203). In hereditary hemochromatosis (HH), however, this regulatory mechanism seems to be impaired; despite iron overload, elevated amounts of iron are absorbed from the diet and lead to accumulation of excess iron in internal organs, resulting in organ dysfunction and failure. The molecular mechanisms by which the intestine responds to alterations in body iron requirements is poorly understood. In this context, hepcidin, a recently identified mammalian peptide (Krause et al. (2000) FEBS Lett 489, 147-150; Park et al. (2001) J Biol Chem 276, 7806-7810), is predicted as a key signaling component regulating iron homeostasis (Philpott C. C. (2002) Hepatology 35, 993-1001; Nicolas et al. (2002) Proc Natl Acad Sci USA 99, 4596-4601)
Hepcidin is a small cysteine-rich peptide predominantly produced in the liver. This molecule regulates the absorption of iron in the intestine and inhibits release of iron from macrophages. Hepcidin was initially isolated as an amino acid (aa) peptide in human plasma and urine exhibiting antimicrobial activity (Krause et al. (2000) FEBS Lett 489, 147-150; Park et al. (2001) J Biol Chem 276, 7806-7810). Hepcidin cDNAs encoding an 83 aa precursor in mice and an 84 aa precursor in rat and man, including a putative 24 aa signal peptide, were subsequently identified searching for liver-specific genes that were regulated by iron (Pigeon et al. (2001) J Biol Chem 276, 7811-7819). A cDNA structure for human hepcidin suggests that it is translated as an 84 amino acid prepropeptide that is amino terminally processed to a 60 amino acid residue prohepcidin peptide, which is further processed into a 25 amino acid hepcidin peptide. (Park et al. (2001).
Hepcidin expression is abolished in mice exhibiting iron-overload due to the targeted disruption of upstream stimulatory factor 2 (Usf2) gene resembling the same phenotype as found in hfe−/− mice (Nicolas G, et al (2001) Proc Natl Acad Sci USA 98, 8780-8785), leading to the conclusion that this peptide plays a pivotal role in iron metabolism. In contrast, overexpression of hepcidin was shown to result in severe iron deficiency anemia in transgenic mice (Nicolas et al. (2002) Proc Natl Acad Sci USA 99, 4596-4601), indicating that hepcidin is a central regulator of iron homeostasis. Moreover, recent studies have shown that liver hepcidin expression is decreased in the hfe knockout mouse (Ahmad et al. (2002) Blood Cells Mci Dis 29, 361-366) and mutations in the hepcidin peptide are associated with severe juvenile hemochromatosis (Roetto et al. (2003) Nat Genet 33, 21-22), opening new perspectives in understanding of the molecular pathogenesis of iron overload. However, the mechanism by which hepcidin balances the body iron stores or adjusts the dietary iron absorption in physiologic and pathologic conditions still remains to be identified.
In this respect, the cellular localization of this peptide and its regulation in various iron states are of major importance in the study of hepcidin function. Although Northern blot analysis of human and mouse hepcidin mRNA levels in various organs revealed that hepcidin is predominantly expressed in liver (Krause et al. (2000) FEBS Lett 489, 147-150; Park et al. (2001) J Biol Chem 276, 7806-7810; Nicolas et al. (2002) Proc Natl Acad Sci USA 99, 4596-4601), no data exists on the cellular localization of this peptide.